LJH685

RSK1/Ribosomal S6 Kinase (IC50 = 6 nM); RSK1 (IC50 = 5 nM); RSK1 (IC50 = 4 nM)

LJH685（0.01-100 μM；72 小时）抑制软琼脂中 MDA-MB-231 和 H358 细胞生长的有效 EC50 值分别为 0.73 和 0.79 μM。 LJH685（0.1-10 μM；4 小时）在亚微摩尔浓度下有效，在较高浓度下几乎完全抑制 YB1 磷酸化[1]。

RSK抑制剂LJH685通过干扰YB-1-KLF5轴在体内抑制BLBC细胞的肿瘤发生。我们的数据表明，YB-1在多个水平上积极调节KLF5，促进BLBC的进展。新型RSK2-YB-1-KLF5-KRT16/Ly6D轴为BLBC提供了候选诊断标志物和治疗靶点。[2]
为了测试这一点，我们在HCC1806原位异种移植物小鼠模型中评估了LJH685的抗肿瘤作用。正如预期的那样，我们观察到LJH685给药后肿瘤生长受到显著抑制（图7H，I），尽管该化合物在水中的溶解度低，在血液中的半衰期短。LJH685治疗的小鼠没有明显的体重减轻，表明其毒性是可以接受的（补充图S8E）。此外，我们证明LJH685降低了YB-1 pS102、KLF5、KRT16、Ly6D和Cyclin D1的表达，但增加了异种移植物肿瘤组织中切割的PARP1（补充图S8F）。总之，LJH685在体内有效抑制HCC1806细胞生长，因此是治疗BLBC的潜在药物。[2]

重组全长 RSK 蛋白用于测量 RSK 亚型 1、2 和 3（PV4049、PV4051 和 PV3846）的酶活性。在 ATP 存在下，RSK1 (1 nmol/L)、RSK2 (0.1 nmol/L) 或 RSK3 (1 nmol/L) 可以磷酸化 200 nmol/L 肽底物 (生物素-AGAGRSRHSSYPAGT-OH)，浓度等于每种酶的 ATP 的 Km（RSK1，5 μmol/L；RSK2，20 μmol/L；RSK3，10 μmol/L）以及 RSK 抑制剂的适当稀释度。

通过在含有细胞生长培养基的 96 孔组织培养处理板上每孔接种 1000 个细胞，可以测量附加条件下的细胞生长。 72小时后，根据制造商的说明添加CellTiter Glo试剂来评估细胞生长。将适当稀释的化合物添加到细胞上方的培养基中。

Administration of LJH685 in tumour burden mice[2]
HCC1806 cells (6 × 105) were injected subcutaneously into both the left and right mammary fat pads of twelve female nude mice (6–7-week-old). After seven days, the tumour volume and weight of mice were measured, and mice were randomly distributed into two groups. The mice were then treated with LJH685 or NC by intraperitoneal injection daily. The tumour volume and mouse weight were measured every other day. The mice were sacrificed on day 21, and the tumours were harvested and weighed. LJH685 was prepared by adding each of the following solvents in sequential order; 10% DMSO, 40% PEG300, 5% Tween-80, and 45% saline. The drug solution was freshly prepared to avoid freeze thawing that could cause drug precipitation.

[1]. Mol Cancer Res. 2014 May;12(5):803-12.

[2]. YB-1 is a positive regulator of KLF5 transcription factor in basal-like breast cancer. Cell Death & Differentiation volume 29, pages1283–1295 (2022)

The p90 ribosomal S6 kinase (RSK) family of serine/threonine kinases is expressed in a variety of cancers and its substrate phosphorylation has been implicated in direct regulation of cell survival, proliferation, and cell polarity. This study characterizes and presents the most selective and potent RSK inhibitors known to date, LJH685 and LJI308. Structural analysis confirms binding of LJH685 to the RSK2 N-terminal kinase ATP-binding site and reveals that the inhibitor adopts an unusual nonplanar conformation that explains its excellent selectivity for RSK family kinases. LJH685 and LJI308 efficiently inhibit RSK activity in vitro and in cells. Furthermore, cellular inhibition of RSK and its phosphorylation of YB1 on Ser102 correlate closely with inhibition of cell growth, but only in an anchorage-independent growth setting, and in a subset of examined cell lines. Thus, RSK inhibition reveals dynamic functional responses among the inhibitor-sensitive cell lines, underscoring the heterogeneous nature of RSK dependence in cancer.[1]

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Y-box binding protein 1 (YB-1) is a well-known oncogene highly expressed in various cancers, including basal-like breast cancer (BLBC). Beyond its role as a transcription factor, YB-1 is newly defined as an epigenetic regulator involving RNA 5-methylcytosine. However, its specific targets and pro-cancer functions are poorly defined. Here, based on clinical database, we demonstrate a positive correlation between Kruppel-like factor 5 (KLF5) and YB-1 expression in breast cancer patients, but a negative correlation with that of Dachshund homolog 1 (DACH1). Mechanistically, YB-1 enhances KLF5 expression not only through transcriptional activation that can be inhibited by DACH1, but also by stabilizing KLF5 mRNA in a RNA 5-methylcytosine modification-dependent manner. Additionally, ribosomal S6 kinase 2 (RSK2) mediated YB-1 phosphorylation at Ser102 promotes YB-1/KLF5 transcriptional complex formation, which co-regulates the expression of BLBC specific genes, Keratin 16 (KRT16) and lymphocyte antigen 6 family member D (Ly6D), to promote cancer cell proliferation. The RSK inhibitor, LJH685, suppressed BLBC cell tumourigenesis in vivo by disturbing YB-1-KLF5 axis. Our data suggest that YB-1 positively regulates KLF5 at multiple levels to promote BLBC progression. The novel RSK2-YB-1-KLF5-KRT16/Ly6D axis provides candidate diagnostic markers and therapeutic targets for BLBC.[2]

C22H21F2N3O

381.42

381.165

C, 69.28; H, 5.55; F, 9.96; N, 11.02; O, 4.19

1627710-50-2

1627710-50-2

73010393

White to off-white solid powder

1.3±0.1 g/cm3

481.1±45.0 °C at 760 mmHg

244.7±28.7 °C

0.0±1.3 mmHg at 25°C

1.601

3.31

39.6Ų

1

6

3

28

482

0

C1=C(C2C(C3C=CC(N4CCN(CC4)C)=CC=3)=CC=NC=2)C=C(C(O)=C1F)F

IKUFKDGKRLMXEX-UHFFFAOYSA-N

InChI=1S/C22H21F2N3O/c1-26-8-10-27(11-9-26)17-4-2-15(3-5-17)18-6-7-25-14-19(18)16-12-20(23)22(28)21(24)13-16/h2-7,12-14,28H,8-11H2,1H3

2,6-difluoro-4-[4-[4-(4-methylpiperazin-1-yl)phenyl]pyridin-3-yl]phenol

LJH685; LJH685; LJH685; NVP LJH685; NVP-LJH685;NVPLJH685; NVP LJH 685; 2,6-difluoro-4-[4-[4-(4-methylpiperazin-1-yl)phenyl]pyridin-3-yl]phenol; 2,6-Difluoro-4-{4-[4-(4-Methylpiperazin-1-Yl)phenyl]pyridin-3-Yl}phenol; CHEMBL3604793; 27CZQ807C1; NVP-LJH-685

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: ~20 mg/mL (~52.4 mM)
Ethanol: ~10mg/mL (~26.2 mM)

配方 1 中的溶解度: ≥ 1 mg/mL (2.62 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 10.0 mg/mL澄清DMSO储备液加入400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 1 mg/mL (2.62 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 10.0 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 1 mg/mL (2.62 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 10.0 mg/mL 澄清 DMSO 储备液加入900 μL 玉米油中，混合均匀。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。